Every year, there are more than 45,000 new cases of squamous cell carcinoma of the head and neck (SCCHN) in the United States and 500,000 new cases woridwide. While most of the cases of SCCHN result from alcohol consumption and/or tobacco use, about 30 percent of SCCHN contain human papillomavirus (HPV) type 16 DNA. HPVs are common wart-causing viruses that infect the basal lamina of the skin or mucous membranes. The HPV-positive subset appears to be increasing in the past 10 years, often occurs in individuals without the risk factors of alcohol and tobacco. Interestingly, the presence of HPV DNA correlates with decreased metastasis, increased treatment response to chemotherapy and radiation, and an approximately 60% decrease in mortality rate from this cancer. Most cases of HPV-positive SCCHN overexpress the viral oncogenes E6 and E7, which inactivate cellular tumor suppressors, including p53 and pRB. Micro (mi) RNAs are a recently-discovered class of gene regulators that most commonly function as negative regulators of gene expression. MiRNAs are ~22nt single-stranded RNA molecules that target messenger RNAs (mRNAs) for translational repression or mRNA cleavage. MiRNA targets include genes involved in development and cell cycle control. We have shown that HPV-positive and HPV-negative SCCHN have different cellular miRNA expression profiles from each other and from normal oral tissue. We hypothesize the difference in cellular miRNA expression will lead to alterations in the expression of genes targeted by such miRNAs, thus affecting pathways involved in the development of HPV-positive and HPV- negative SCCHN. We have shown that miR-363 is specifically upregulated in HPV-positive SCCHN cell lines and tissue samples compared to the HPV-negative counterpart. The specific aims of this proposal are: (i) to investigate the role of the HPV-16 oncogenes in the differential expression of miR-363. Immortalized normal oral kerotinocytes expressing the HPV-16 oncogenes E6 and E7, will be utilized for experimental methods, and if one or both of the oncogenes play a role in upregulation of miR-363 in HPV-positive SCCHN, the specific domain of the oncogene responsible will be determined using mutafional analysis; (ii) to identify the gene target(s) of miR-363. Possible targets will be identified by utilizing gene expression array data generated in our lab, computafional analysis, and quantitative Real Time RT-PCR. Public Health Relevance: This study will aid in understanding the pathways involved in HPV-associated oral carcinogenesis. Exploring the differences in miRNA expression in HPV-positive and HPV-negative SCCHN could lead to better diagnostics, treatments, and cures for oropharyngeal cancers.